A–CFLAG‐tagged CYLD or catalytically inactive FLAG‐CYLD (C598A), respectively, was stably expressed in CYLD−/− MEFs, followed by infection with control retrovirus, or retrovirus encoding SPATA2, and purified by FLAG‐IP. Purified CYLD or the inactive mutant was added to recombinant diubiquitin for 0, 20, or 40 min at 37°C as indicated. After electrophoresis, the gel was cut and the lower part was silver‐stained to visualize mono‐ and diubiquitin, while the upper part was transferred to a membrane and probed for FLAG and V5. In (A), the experiment was done with M1‐linked diubiquitin substrate; in (B), K63‐linked diubiquitin substrate was subjected to the assay; and in (C), K48‐linked diubiquitin substrate was analyzed.
